A major drawback of human mesenchymal stem cell (hMSC) usage in cell based therapies is the difficulty of achieving sufficient cell numbers for therapeutic purposes.
Current strategies which include use of extra cellular matrix (ECM) substrates and using fibroblast growth factor 2 (FGF2) give higher cell counts but lead to increase in differentiated progenitors in the cell populations.
The low numbers of hMSCs, where it can be as low as 0.01% to 0.0001% of bone marrow mononuclear cells, hinders their widespread usage. In addition, current ex vivo expansion strategies will lead to loss of multipotentiality leading to enough cells for transplant with less quality and function.
Brickman et al. (Glycobiology Vo. 8 No. 5 pp. 463-471, 1998) describe an heparan sulphate called HS2 reported to be capable of interacting with FGF2. HS2 is obtainable from heparan proteoglycans of murine cells at embryonic day 10 as described by Brickman (supra). HS2 has been described as having a molecular weight of approximately 25 kDa and thus, assuming an average molecular mass of 400 Da per disaccharide, consists of about 60 disaccharides. The disaccharide composition of HS2 is set forth in Brickman et al. (Glycobiology Vo. 8 No. 5 pp. 463-471, 1998), WO2010/011185, which is herein incorporated by reference in its entirety. The nitrous acid and heparan lyase digestion profiles of HS2 are shown in FIGS. 29 and 30.
Maccarana et al (Minimal Sequence in Heparin/Heparan Sulfate Required for Binding of Basic Fibroblast Growth Factor. The Journal of Biological Chemistry. Vol. 268, No. 32, Issue 15, pp23898-23905, 1993) describes experiments investigating the binding of FGF-2 by several small oligosaccharides generated from heparin or HS from human aorta. One octasaccharide fraction (B2) was used to ascribe a structure to the octasaccharide, which the authors called HS-8. It should be noted that this is not the HS-8 of the present invention and the nomenclature is entirely coincidental.
Heparin from pig intestinal mucosa, two samples of selectively O-desulfated heparin, one sample generated by preferential 6-O-desulfation together with N-desulfation of the starting material followed by re-N-sulfation, another sample obtained by selective 2-O-desulfation under alkaline conditions, a low sulphated HS isolated from human aorta, and HS from bovine kidney were used to generate low chain length oligosaccharides of even or odd number.
Even number oligosaccharides were generated from heparin by depolymerisation through partial deaminative cleavage with nitrous acid and the resulting 2,4-anhydro-D-mannose residues were reduced with NAB3H4. Labeled oligosaccharides were separated to generate even numbered species from 4-14 saccharides and a fraction containing 20-24 saccharides. The selectively 6-O-desulfated heparin was similarly treated to yield 4- to 12-saccharides. The isolated and desalted oligosaccharides were subjected to mild acid treatment. Odd numbered heparin oligosaccharides were obtained by further treatment of the 20-24-meric saccharides with heparinase I.
4-14-meric oligosaccharides were generated from human aorta HS by a different strategy involving cleavage at sites occupied by N-acetylated GlcN units. Samples were N-deacetylated and then deaminated with nitrous acid. This treatment leads to deamination of unsubstituted GlcN units and cleavage of glucosaminidic linkages whereas N-sulfated GlcN units remain intact. The products include GlcA-[1-3H]aManR disaccharides (derived from (-GlcNAc)-(GlcA-GlcNac)n-sequences) and GlcA-GlcNSO3-HexA)n-[1-3H]aManR oligosaccharides (derived from (-GlcNac)-GlcA-(GlcNSO3-HexA)n-GlcNac-sequences).
The oligosaccharides generated by these treatments were both short and chemically modified by the process of their preparation, which distinguishes them from the HS of the present invention.